FIELD OF THE INVENTION
The invention describes a method for the quantitative determination of plasmin-.alpha.2-antiplasmin complexes as well as the use of this method as a mean of determining changes in the fibrinolytic system.
Introduction:
.alpha.2-antiplasmin is the most important plasmin inhibitor. Its rapid reaction with plasmin results in the formation of an inactive complex composed of one molecule of each component. Two steps are involved in this process: first, a reversible complex is formed between the lysine-binding site of plasmin and complementary sites on the carboxy terminal end of the .alpha.2-antiplasmin molecule. In a second step, an irreversible complex is generated associated with the cleavage of a peptide-bond in the inhibitor. During activation of plasminogen to plasmin an equilibrium exists between formation of plasmin-.alpha.2-antiplasmin complexes, occurring preferentially in the fluid phase and binding and action of plasmin on the fibrin surface. Bound to fibrin, plasmin is protected against inhibition by .alpha.2-antiplasmin. However, whenever fibrin is completely dissolved the plasmin liberated from the fibrin surface is immediately complexed by .alpha.2-antiplasmin.
Whenever fibrin forms in circulation, this process will be accompanied by activation of the fibrinolytic system because of the well known effects of fibrin on tissue plasminogen activator (t-PA). Plasmin generated thereby will in part become complexed with .alpha.2-antiplasmin leading to increased levels of plasmin-.alpha.2-antiplasmin complexes. Such increased levels of PAP-complexes have therefore been found in many circumstances in which fibrin formation is increased as in thrombophilia, hypercoagulability, disseminated intravascular coagulation, endotoxin shock, leukemia, liver diseases, nephrotic syndrome or after major surgery. Even in plasma after venous occlusion in most cases increased plasmin-.alpha.2-antiplasmin levels have been found consistent with increased fibrin formation and increased levels of tissue plasminogen activator in the venous occlusion plasma.
It is to be expected that during formation of serum a time dependent generation of PAP complexes occurs. The amount of PAP complexes generated in serum can be expected to be dependent on the amount of other plasminogen activators present in the respective environement as well as on plasminogen activator inhibitors, receptors and binding proteins.
While in all cases mentioned above only a limited increase of PAP levels is observed, thrombolytic therapy leads to extensive activation of the fibrinolytic system to a massive plasmin formation in the fluid phase and maximal PAP complex formation. Especially nonfibrin specific plasminogen activators such as streptokinase or urokinase can cause complete consumption of plasmin inhibitors resulting in an increased bleeding tendency due to plasminemia. Therefore the plasmin activity is no longer restricted to its specific substrate fibrin but can extend to nonspecific substrates as fibrinogen and other coagulation factors. Therefore, determination of plasmin-.alpha.2-antiplasmin complexes on the one hand can serve as indicative for general plasminemia during hyperfibrinolytic states with fibrinogen and .alpha.2-antiplasmin consumption and possible bleeding tendency; on the other hand, slightly increased levels of plasmin-.alpha.2-antiplasmin complexes are indicative for ongoing thrombus formation and thrombus dissolution as in the case of thrombophilia and PAP complexes in serum, respectively and are dependent on the total fibrinolytic potential.
Methods Available for Determination of PAP:
To determine PAP complexes several test methods have been published including two dimensional immunelectrophoresis, latex assay, RIA and more recently ELISA systems. Initially a latex agglutination assay for determination of PAP complexes was introduced by Plow et al. with rather low sensitivity. Then a two dimensional electrophoresis was described using the different mobility of free and complexed .alpha.2-antiplasmin. This method was still not sensitive enough and rather time consuming and not applicable for a larger number of samples. Employing polyclonal antisera raised against plasmin B-chain .alpha.2-antiplasmin complexes a RIA was developed by Wiman et al. thereby avoiding recognition of intact plasminogen or plasmin. But intact .alpha.2-antiplasmin would still be detected together with the PAP complex, though to a much lesser extent.
A great advance was the development of the double sandwich technique used in the ELISA systems. Several methods have been developed since either utilising an antibody against antiplasmin as a catching antibody and an antibody against the enzyme as detecting antibody or vice versa. Harpel et al. first published a sensitive assay with a polyclonal antibody against .alpha.2-antiplasmin as catching antibody and POX-labeled Fab-fragments against plasminogen as detecting system, whereby the Fab- fragments are preferred to whole antibodies to reduce unspecific binding of plasminogen and other plasma proteins to immuno-globulins. Other ELISA-systems were described by Holvoet et al. and Mimuro et al. employing either two monoclonal antibodies against either part of the complex or one polyclonal and one monoclonal antibody. Most recently a liposome immune lysis assay (LILA) was introduced by Hasada et al. The ELISA or LILA test systems described up to now have, however, certain limitations because of interference of the abundant amounts of uncomplexed .alpha.2-antiplasmin or plasminogen with the rather low concentrations of the PAP complex. All methods described above have, however, the disadvantage of utilising antibodies recognising the complexes as well as the native uncomplexed molecules.
In DE-A1-41 15 993 A method is disclosed for the production of a monoclonal antibody BMA PAP6 and the respective hybridoma cell line (BW PAP6). Said antibody has a specific affinity for plasmin-anti-plasmin complexes and no or very little affinity to the separate components of this complex. This antibody was obtained by immunizing with a plasmin-anti-plasmin complex cleaved by treatment with ammonia. Also, this antibody is only functionally characterized by means of an ELISA but not utilizing a Western Blot